Bv421 anti cd39

PBMC or cultured T cells were stained for 30 min at 4°C with following fluorochrome-conjugated Abs: APC-Cy7-anti-CD3, v500-anti-CD4, PE-Cy5-anti-CD25, APC-anti-CD25, PE-Cy7-anti-CD45RA, BV421-anti-CD161, (six from BD Bioscience), FITC-anti-GARP (Enzo Life Science, Farmingdale, NY), PE-Cy7-anti-GITR, APC-Cy7-anti-CD73, BV421-anti-CD39, PerCP-Cy5.5-anti-CTLA-4, PE-Cy7-anti-CXCR3 (five from BioLegend, San Diego, CA), PE-anti-IL-1RI, APC-anti-IL-1RII, and FITC-anti-IL-1RII (three from R and D systems). For intracellular cytokine staining (ICS), cultured T cells were re-stimulated for 6 hr with PMA (50 ng/ml) and ionomycin (1 μg/ml) in the presence of BFA for last 4 hr, followed by staining with PE-anti-IL-1RI and FITC-anti-IL-1RII Abs. The stained cells were fixed and permeabilized using Fix/Perm buffer (BioLegend), followed by staining with anti-IL-17A, anti-IFN-γ (both from eBioscience), and anti-Foxp3 (Biolegend) Abs. The cells were acquired using a BD LSRFortessa (BD Bioscience) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Anti-CD14 conjugated to violet blue was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). FITC-anti-CD4, PE Cy5-anti-CD11c, PE-anti-CD25, BV650-anti-CD25, APC-anti-CD45RA, BV711-anti-CD15s, PE-anti-CD45RA, PC5-anti-CD28, and PE anti-CTLA-4 were obtained from BD Biosciences (Mountain View, CA, USA). PE CY7 anti-CD127, BV421-anti CCR7, PE-anti-CD59, and BV510-anti-CD27 were purchased from Sony Biotechnology (San Jose, CA, USA); PE-FOXP3, APC-FOXP3, and eF450-Helios were supplied by eBiosciences (San Diego, CA, USA); PC5 anti-ICOS, APC-anti-PD1, BV421-anti-CD39, APC-anti-GITR, and BV421-anti LAG3 were purchased from Biolegend (San Diego, CA, USA); and staphylococcal enterotoxin E (SEE) was purchased from Toxin Technology Inc.; and CellTrace carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA). The anti-CD3 (OKT3) used for some proliferation assays was purchased from Biolegend (San Diego, CA, USA).